Characterization of Arabidopsis thaliana Coq9 in the CoQ Biosynthetic Pathway

Coenzyme Q, also known as ubiquinone, is a fat-soluble isoprene quinone that serves as a cofactor for numerous enzymes across all domains of life. However, the biosynthetic pathway for this important molecule in plants has been examined in only a limited number of studies. In yeast and mammals, Coq9, an isoprenoid-lipid-binding protein, is essential for CoQ biosynthesis. Previous studies showed that Arabidopsis thaliana Coq9 failed to complement the fission yeast Schizosaccharomyces pombe coq9 null mutant, and its function in plants remains unknown. In this study, we demonstrated that expression of Arabidopsis Coq9 rescued the growth of a yeast temperature-sensitive coq9 mutant and increased CoQ content. Phylogenetic analysis revealed that Coq9 is widely present in green plants. Green fluorescent protein (GFP) fusion experiments showed that Arabidopsis Coq9 is targeted to mitochondria. Disruption of the Coq9 gene in Arabidopsis results in lower amounts of CoQ. Our work suggests that plant Coq9 is required for efficient CoQ biosynthesis. These findings provide new insights into the evolution of CoQ biosynthesis in plants. The identification of Coq9 as a key player in CoQ biosynthesis in plants opens up new avenues for understanding the regulation of this important metabolic pathway.


Introduction
Coenzyme Q (CoQ, ubiquinone) is a conserved redox-active, hydrophobic lipid that exists in most biological membranes. It serves as an electron transporter in the mitochondrial respiratory chain and accepts electrons from several other mitochondrial inner-membrane dehydrogenases [1]. Beyond its roles in mitochondria, the extramitochondrial CoQ has widespread cellular functions, including combatting ferroptosis and modulating membrane structure dynamics, as discussed in recent reviews [2].
CoQ is composed of a benzoquinone head group and a polyisoprenoid tail with a length that varies among species. The aromatic ring precursor and the polyprenyldiphosphate tail precursor are synthesized in the cytosol and transported into the mitochondrial matrix. The yeast Saccharomyces cerevisiae has been used as an experimental model to study the eukaryotic CoQ biosynthesis pathway [3,4]. Currently, at least six enzymes (Coq1, Coq2, Coq3, Coq5, Coq6, and Coq7) and four auxiliary proteins (Coq4, Coq8, Coq9, and Coq11) are known to participate in CoQ biosynthesis in yeast mitochondria [2,3,5]. The genes responsible for CoQ biosynthesis in S. cerevisiae and humans are indicated in capital letters, while the corresponding proteins are denoted in uppercase letters for humans, with only the first letter capitalized in S. cerevisiae. In Arabidopsis, the genes and proteins are written with only the first letter capitalized, for instance, human gene COQ9, human protein COQ9, yeast gene COQ9, yeast protein Coq9, Arabidopsis gene Coq9, and Arabidopsis

Beta-Glucuronidase (GUS) Reporter Assay
Promoter regions of AtCoq9 (2067 bp upstream of the start codon) were amplified by polymerase chain reaction (PCR) from Arabidopsis Col-0 genomic DNA with highfidelity DNA polymerase (FastPfu, TransGen, Beijing, China) using the primers as listed (Supplementary Table S1). The amplified fragments were cloned into vector pDONR207 using Gateway technology. Then, the promoter region was introduced into the destination vector pGWB533. Afterwards, the clone was introduced into Agrobacterium tumefaciens strain GV3101 (purchased from WEIDI, Shanghai, China) and transformed into Arabidopsis through floral dipping [28].

Subcellular Localization
Full-length coding regions of AtCoq9 were amplified by PCR from Arabidopsis complementary DNAs (cDNAs) with high-fidelity DNA polymerase. Subsequently, the PCR product was cloned into the destination vector pGWB505 (a gift from Dr. Ping Xu). After sequence verification, the plasmid was transformed into A. tumefaciens strain GV3101 (pSoup-p19) (purchased from WEIDI).
Single colonies for each construct were resuspended in Yeast Extract Peptone medium (10 g/L yeast extract, 10 g/L Bacto peptone, and 5 g/L NaCl); supplied with 20 mg/L rifampicin, 40 mg/L gentamycin, and 75 mg/L spectinomycin; and cultured at 28 • C for 1 day. Bacterial cultures were centrifuged at 8000× g for 5 min. Cell pellets were washed with an induction buffer (10 mM MES, pH 5.7, 10 mM MgCl 2 , and 200 µM acetosyringone), and then resuspended in the induction buffer to a final OD 600 of 0.3, which was determined using a NanoCrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Before injection, strains were incubated at room temperature for 3 h. The agrobacteria were then injected into the abaxial side of N. benthamiana leaves with a 1 mL needleless syringe. Vector CD3-991 (mitochondria targeted mCherry) was coinfiltrated with GFP constructs [30].
The transformed plants were grown in the greenhouse for 3 days. Images were captured using an Olympus FV10i confocal laser scanning microscope. Excitation wavelengths were 473 nm for GFP and 580 nm for mCherry. Emission fluorescence was captured by 490 to 540 nm for GFP and 610 nm for mCherry.

Quantitative Reverse-Transcription PCR
Tissue samples were collected and frozen in liquid nitrogen. Total RNA from 7-day-old seedlings, 14-day-old seedlings, 4-week-old rosette leaves, stems and stem leaves of 5-weekold plants, flowers, 3-day-old siliques, and 12-day-old siliques was isolated using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. Total RNA from mature seeds was isolated with the RNAprep Pure Plant Plus Kit (TIANGEN, Beijing, China). RNA was reverse-transcribed with the PrimeScript RT reagent Kit with the gDNA Eraser (Takara, Dalian, China). qRT-PCR was performed in triplicate on Mastercycler ep Realplex2 (Eppendorf, Hamburg, Germany), using TB Green Premix Ex Taq (Takara). PCR cycling was performed as follows: 30 s at 95 • C, followed by 40 cycles of 10 s at 95 • C, 15 s at 60 • C, and 20 s at 72 • C. To confirm the specificity of primer amplification, a melting curve was generated by gradually increasing the temperature from 75 • C to 95 • C in increments of 0.5 • C. Three technical replicates were performed for each tissue sample. The 2 −∆∆Ct method was used to determine the relative expression. The Arabidopsis gene PP2AA3 (At1g13320) was used as an endogenous control [31]. The primers for qRT-PCR analysis are presented in Supplementary Table S1.

Yeast Strain and Complementation Assays
Saccharomyces cerevisiae strain BY4742 was used in this study as control strain. ∆coq9 (Y14150) was purchased from Euroscarf. Yeast was transformed using the Frozen-EZ Yeast Transformation kit (ZYMO, Irvine, CA, USA) according to the manufacturer's protocol and grown at 30 • C on SD-Leu-His agar media (0.67% BD Difco yeast nitrogen base with ammonium sulfate, 0.62 g/L Clontech Dropout supplement -His/-Leu/-Trp, 20 mg/L tryptophan, 2% dextrose and 2% Bacto agar).
The yeast COQ9 gene was amplified from the genomic DNA of BY4742 by PCR. The ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China) was used to insert PCR amplicons into the XbaI-and BamHI-linearized pRS315 vector. The plasmid TS19 was generated according to [15]. Site-directed mutagenesis (E55G, R107G, and Q256L) was performed using the fully assembled ScCoq9-pRS315 plasmid as a template, with specific mutations generated by PCR. Full-length coding regions of AtCoq9 were amplified by PCR from Arabidopsis cDNAs and cloned into pRS423 controlled by the S. cerevisiae COQ8 promoter and terminator. Human COQ9 gene sequence was synthesized (GenScript) and cloned into pRS423. The mitochondrial targeting sequence of yeast Coq3 was fused with AtCoq9 and human COQ9.

Analysis of CoQ Contents
Yeast cells were grown at 25 • C or 30 • C on SD-Leu-His liquid medium with 2% glucose until OD 600 reached~1. Cells with a quantity totaling 10 mL were centrifuged, and the pellet mass was determined. Glass beads (0.5 mm diameter, 100 µL), 1 mL of isopropanol, and internal standard CoQ 4 (0.1 mg/L) were added to the cell pellets (50 mg wet weight). Samples were vortexed (2 min) and extracted in a sonicator bath for 30 min. After centrifugation at 12,000 rpm for 10 min, the pellets were extracted with 1 mL of isopropanol in a sonicator bath for 30 min and centrifuged at 12,000 rpm for 10 min. The supernatant from two extractions were combined for LC-MS analysis.
For Arabidopsis, samples were harvested, frozen in liquid nitrogen, and freeze-dried. Ten milligrams of freeze-dried samples were extracted with 1 mL of isopropanol in a sonicator bath for 60 min.

Phylogenetic Analysis of Plant Coq9
To examine the distribution of Coq9 in green plants, we screened the genomes of 62 representative species of Viridiplantae via BLAST, using yeast Coq9 and human COQ9 as query sequences. A phylogenetic tree based on sequences from two chlorophytes, three mosses, one liverwort, one lycophyte, one fern, one conifer, and fifty-three angiosperms was inferred using the maximum-likelihood method ( Figure 1). To root the tree, human COQ9 and yeast Coq9 were included as an outgroup. Coq9 was identified in all taxa investigated. The phylogenetic tree of Coq9s complies with the branches of Viridiplantae.

Phylogenetic Analysis of Plant Coq9
To examine the distribution of Coq9 in green plants, we screened the genomes of 62 representative species of Viridiplantae via BLAST, using yeast Coq9 and human COQ9 as query sequences. A phylogenetic tree based on sequences from two chlorophytes, three mosses, one liverwort, one lycophyte, one fern, one conifer, and fifty-three angiosperms was inferred using the maximum-likelihood method (Figure 1). To root the tree, human COQ9 and yeast Coq9 were included as an outgroup. Coq9 was identified in all taxa investigated. The phylogenetic tree of Coq9s complies with the branches of Viridiplantae.  Coq9 is widely distributed in green plants. Amino acid sequences from two chlorophytes, three mosses, one liverwort, one lycophyte, one fern, one conifer, and fifty-three angiosperms were aligned using MAFFT. Maximum-likelihood phylogenetic tree was constructed using IQ-TREE under the JTT + G4 model selected by ModelFinder. The tree was rooted using human COQ9 (NP_064708.1) and yeast Coq9 (QHB10353.1) as an outgroup. Black circles are used to represent bootstrap values over 90%, and grey circles are used represent bootstrap values between 70% and 90%.
Further analysis of the numbers of Coq9 genes showed that diploid species, such as Arabidopsis, Solanum lycopersicum, Oryza sativa, Zea mays, and Sorghum bicolor, retained one copy, the palaeopolyploid Glycine max contained two copies, and the hexaploid Triticum aestivum had three copies. These data suggest that Coq9 was widely present in Viridiplantae and did not undergo multiple duplications.

Expression Patterns of AtCoq9
To profile the expression patterns of AtCoq9, quantitative reverse-transcription PCR (qRT-PCR) was performed using Arabidopsis RNAs from the seedlings, rosettes, stems, stem leaves, flowers, siliques, and mature seeds. The AtCoq9 gene was widely expressed, with the highest levels in mature seeds (Figure 2A), where the CoQ contents were also most abundant [7]. To further examine the spatiotemporal expression of AtCoq9, transgenic Arabidopsis plants carrying a ProAtcoq9::GUS construct were generated. Three independent transgenic lines were selected for GUS staining ( Figure 2B). Ubiquitous GUS activity was observed in 7-day-old seedlings and 14-day-old seedlings. In four-week-old plants, a weak signal could be observed in young leaves, while strong expression was found in old leaves. In inflorescence, AtCoq9 was again widely expressed. In summary, AtCoq9 is ubiquitously expressed throughout plant development.
bootstrap values over 90%, and grey circles are used represent bootstrap values between 70% and 90%.
Further analysis of the numbers of Coq9 genes showed that diploid species, such as Arabidopsis, Solanum lycopersicum, Oryza sativa, Zea mays, and Sorghum bicolor, retained one copy, the palaeopolyploid Glycine max contained two copies, and the hexaploid Triticum aestivum had three copies. These data suggest that Coq9 was widely present in Viridiplantae and did not undergo multiple duplications.

Expression Patterns of AtCoq9
To profile the expression patterns of AtCoq9, quantitative reverse-transcription PCR (qRT-PCR) was performed using Arabidopsis RNAs from the seedlings, rosettes, stems, stem leaves, flowers, siliques, and mature seeds. The AtCoq9 gene was widely expressed, with the highest levels in mature seeds (Figure 2A), where the CoQ contents were also most abundant [7]. To further examine the spatiotemporal expression of AtCoq9, transgenic Arabidopsis plants carrying a ProAtcoq9::GUS construct were generated. Three independent transgenic lines were selected for GUS staining ( Figure 2B). Ubiquitous GUS activity was observed in 7-day-old seedlings and 14-day-old seedlings. In four-week-old plants, a weak signal could be observed in young leaves, while strong expression was found in old leaves. In inflorescence, AtCoq9 was again widely expressed. In summary, AtCoq9 is ubiquitously expressed throughout plant development.

AtCoq9 Is Localized in Mitochondria
AtCoq9 contains the N-terminal mitochondrial targeting sequence, as predicted by TargetP [34]. To examine the subcellular localization of AtCoq9, green fluorescent protein (GFP) was fused to the carboxy terminus of AtCoq9 and transiently expressed in N. ben-

AtCoq9 Is Localized in Mitochondria
AtCoq9 contains the N-terminal mitochondrial targeting sequence, as predicted by TargetP [34]. To examine the subcellular localization of AtCoq9, green fluorescent protein (GFP) was fused to the carboxy terminus of AtCoq9 and transiently expressed in N. benthamiana leaves, along with the mitochondrial mCherry-tagged marker (Figure 3). The signal of GFP fused with AtCoq9 did overlap with the fluorescence of the mCherry mitochondrial marker, thus confirming its predicted mitochondrial localization, which is consistent with previous high-throughput studies [35].

AtCoq9 Rescues the Yeast coq9 Point Mutant
Previous studies showed that expression of either AtCoq9 or human COQ9 did not complement the S. pombe coq9 null mutant [21]. Meanwhile, expression of human COQ9 rescued the S. cerevisiae temperature-sensitive mutant coq9-ts19, which requires the presence of E55G, R107G, and Q256L mutations [18]. Thus, we tested whether expression of AtCoq9 could also rescues a coq9 temperature-sensitive mutant. We transformed the TS19 plasmids containing the yeast COQ9 gene with three mutations into the yeast coq9 null mutant to generate a coq9 temperature-sensitive mutant according to [15]. The coq9-ts19 mutant had decreased CoQ 6 content and failed to grow at the non-permissive temperature of 37 • C [15]. thamiana leaves, along with the mitochondrial mCherry-tagged marker (Figure 3). The signal of GFP fused with AtCoq9 did overlap with the fluorescence of the mCherry mitochondrial marker, thus confirming its predicted mitochondrial localization, which is consistent with previous high-throughput studies [35].

AtCoq9 Rescues the Yeast coq9 Point Mutant
Previous studies showed that expression of either AtCoq9 or human COQ9 did not complement the S. pombe coq9 null mutant [21]. Meanwhile, expression of human COQ9 rescued the S. cerevisiae temperature-sensitive mutant coq9-ts19, which requires the presence of E55G, R107G, and Q256L mutations [18]. Thus, we tested whether expression of AtCoq9 could also rescues a coq9 temperature-sensitive mutant. We transformed the TS19 plasmids containing the yeast COQ9 gene with three mutations into the yeast coq9 null mutant to generate a coq9 temperature-sensitive mutant according to [15]. The coq9-ts19 mutant had decreased CoQ6 content and failed to grow at the non-permissive temperature of 37 °C [15].
Expression of AtCoq9 containing the yeast Coq3 mitochondrial targeting sequence restored the growth of the coq9-ts19 mutant at 37 °C on SD-Leu-His media ( Figure 4A). Analysis of the lipid extract by LC-MS/MS indicated that the presence of AtCoq9 or human COQ9 significantly increased the amount of CoQ6 at both permissive (25 °C) and non-permissive temperatures (37 °C), with AtCoq9 providing noticeably more rescue ( Figure 4B). These results indicate a functional conservation of Coq9 across different organisms. Expression of AtCoq9 containing the yeast Coq3 mitochondrial targeting sequence restored the growth of the coq9-ts19 mutant at 37 • C on SD-Leu-His media ( Figure 4A). Analysis of the lipid extract by LC-MS/MS indicated that the presence of AtCoq9 or human COQ9 significantly increased the amount of CoQ 6 at both permissive (25 • C) and nonpermissive temperatures (37 • C), with AtCoq9 providing noticeably more rescue ( Figure 4B). These results indicate a functional conservation of Coq9 across different organisms.

AtCoq9 Defective Mutants Contained Less CoQ
To study the in vivo function of AtCoq9, we generated AtCoq9-knockout mutants by CRISPR-Cas9-mediated genome editing.gRNA was designed to target the first exon of AtCoq9 ( Figure 5A), and the construct carrying the gRNA and Cas9 protein was transformed in Arabidopsis plants. Two independent knockout alleles were identified in the T1 generation, both of which contained 1 bp insertion and resulted in premature stop codons  HsCOQ9), and TS19 on pRS315 and pRS423-AtCoq9 (TS19 + AtCoq9). Serial dilutions were spotted onto SD-Leu-His or YPG plate media and incubated at either 25 • C or 37 • C. (B) Quantification of CoQ 6 contents. Yeast cells were cultivated in SD-Leu-His liquid media containing 2% glucose and grown overnight at either 25 • C or 37 • C. Lipids were extracted and analyzed by LC-MS. Asterisks indicate significant difference compared to TS19 + vec with * p < 0.05 and ** p < 0.01 (two-tailed Student's t tests). Data are means of three biological replicates ± SE.

AtCoq9 Defective Mutants Contained Less CoQ
To study the in vivo function of AtCoq9, we generated AtCoq9-knockout mutants by CRISPR-Cas9-mediated genome editing.gRNA was designed to target the first exon of AtCoq9 ( Figure 5A), and the construct carrying the gRNA and Cas9 protein was transformed in Arabidopsis plants. Two independent knockout alleles were identified in the T1 generation, both of which contained 1 bp insertion and resulted in premature stop codons for AtCoq9 ( Figure 5B). Homozygous-knockout alleles were obtained in the T2 generation and used for further analyses. In coq9-1 and coq9-3 mutants, the CoQ 9 levels were reduced by 70 to 80% compared to those of wild-type plants ( Figure 5C). However, no phenotypic difference was observed between coq9 mutants and the wild type. Together, these results demonstrate that AtCoq9 is involved in the biosynthesis of CoQ.

Discussion
CoQ is an isoprenoid quinone present in all eukaryotes. The biosynthetic pathway of CoQ in plants has not been fully defined to date [6]. In this study, we found that expression of Arabidopsis Coq9 rescues the yeast coq9 temperature-sensitive mutant. The knockout mutants of Arabidopsis Coq9 contain less CoQ. These findings suggest the evolutionary conservation of Coq9 between plants, yeast, and humans. It also demonstrates that Coq9 is required for efficient CoQ biosynthesis in Arabidopsis.
CoQ is essential for embryogenesis in Arabidopsis. Knockout mutants lacking some of the Coq genes, such as Coq1 [36], Coq2 [37], Coq3 [38], or CoqF [7], are embryo-lethal, and no homozygous mutant plants could be recovered from seeds of heterozygous plants after self-pollination. However, homozygous plants of coq9 knockout mutants were identified, suggesting that Coq9 is not essential for embryo development. In addition, we found that the coq9 knockout mutants still produced small amount of CoQ. The data consistent with the idea that the considerable reduction in CoQ levels in Arabidopsis mutants still enables normal embryo development. Currently, there is relatively little research on the relationship between CoQ metabolism and plant growth and development, and it is worth further study in the future.
In yeast, deletion of the COQ9 gene results in a complete loss of CoQ synthesis and consequently a failure to grow on a nonfermentable carbon source [17]. However, unlike yeast, Arabidopsis lacking Coq9 is not completely CoQ deficient. Although Arabidopsis Coq9 rescued the yeast coq9 temperature-sensitive mutant, it failed to complement the S. pombe coq9 null mutant [21]. Therefore, it is likely that the function of Coq9 may not be fully conserved between yeast and Arabidopsis.
In yeast and mammals, Coq9 is required for the function of Coq7, a carboxylatebridged diiron hydroxylase responsible for catalyzing the penultimate step in CoQ biosynthesis. The position of the Coq9 protein in the CoQ biosynthesis pathway has been reviewed recently [2,5]. However, plants lack Coq7, and C6 hydroxylation is catalyzed by CoqF, a flavin-dependent monooxygenase [7,8]. The functional relationship between Coq9 and CoqF in plants is not yet clear. Moreover, Coq9 has been shown to be present in alpha proteobacterial organisms that do not have Coq7 and has been considered to be an

Discussion
CoQ is an isoprenoid quinone present in all eukaryotes. The biosynthetic pathway of CoQ in plants has not been fully defined to date [6]. In this study, we found that expression of Arabidopsis Coq9 rescues the yeast coq9 temperature-sensitive mutant. The knockout mutants of Arabidopsis Coq9 contain less CoQ. These findings suggest the evolutionary conservation of Coq9 between plants, yeast, and humans. It also demonstrates that Coq9 is required for efficient CoQ biosynthesis in Arabidopsis.
CoQ is essential for embryogenesis in Arabidopsis. Knockout mutants lacking some of the Coq genes, such as Coq1 [36], Coq2 [37], Coq3 [38], or CoqF [7], are embryo-lethal, and no homozygous mutant plants could be recovered from seeds of heterozygous plants after self-pollination. However, homozygous plants of coq9 knockout mutants were identified, suggesting that Coq9 is not essential for embryo development. In addition, we found that the coq9 knockout mutants still produced small amount of CoQ. The data consistent with the idea that the considerable reduction in CoQ levels in Arabidopsis mutants still enables normal embryo development. Currently, there is relatively little research on the relationship between CoQ metabolism and plant growth and development, and it is worth further study in the future.
In yeast, deletion of the COQ9 gene results in a complete loss of CoQ synthesis and consequently a failure to grow on a nonfermentable carbon source [17]. However, unlike yeast, Arabidopsis lacking Coq9 is not completely CoQ deficient. Although Arabidopsis Coq9 rescued the yeast coq9 temperature-sensitive mutant, it failed to complement the S. pombe coq9 null mutant [21]. Therefore, it is likely that the function of Coq9 may not be fully conserved between yeast and Arabidopsis.
In yeast and mammals, Coq9 is required for the function of Coq7, a carboxylatebridged diiron hydroxylase responsible for catalyzing the penultimate step in CoQ biosynthesis. The position of the Coq9 protein in the CoQ biosynthesis pathway has been reviewed recently [2,5]. However, plants lack Coq7, and C6 hydroxylation is catalyzed by CoqF, a flavin-dependent monooxygenase [7,8]. The functional relationship between Coq9 and CoqF in plants is not yet clear. Moreover, Coq9 has been shown to be present in alpha proteobacterial organisms that do not have Coq7 and has been considered to be an ancestral protein of the CoQ biosynthetic pathway [39]. We speculate that Coq9 may perform other roles in addition to assisting the hydroxylase reaction of Coq7. It is interesting to investigate the precise biochemical functions of Coq9 in CoQ biosynthesis in species without Coq7.
In conclusion, this work reveals that Coq9 plays an important role in CoQ biosynthesis in Arabidopsis. It also describes the functional conservation of Coq9 across different organisms. Our findings add to the understanding of the CoQ biosynthetic pathway in plants and provide valuable insights for further exploration of the roles of Coq9. Further studies are needed to elucidate the precise mechanism of Coq9 function in CoQ biosynthesis in plants, as well as to explore the potential applications of CoQ in agriculture and medicine.